miRNAs as Novel Therapeutic Targets and Diagnostic Biomarkers for Parkinsons Disease

ABSTRACT

The disclosure provides pharmaceutical compositions including an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283, or hybridizes under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1 -283. Further provided are methods of diagnosing Parkinson&#39;s Disease (PD) in a subject. The methods may include detecting the level of expression of at least one miRNA of SEQ ID NOs: 1-283 in a biological sample from the subject, and comparing the level of expression in the sample to the level of expression in a reference. Further provided are methods for treating, preventing, or reducing the risk of PD. Kits are also provided.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 61/675,603, filed Jul. 25, 2012, which is incorporated by reference herein in its entirety.

FIELD

This disclosure relates to therapeutic and diagnostic markers for Parkinson's Disease.

INTRODUCTION

MicroRNAs (miRNAs) are small, non-coding, regulatory RNAs of 18-24 nucleotides in length. Mature miRNAs regulate messenger RNAs (mRNAs) of their downstream target genes by base-pairing to their target sites to specify cleavage of the target mRNAs or to destabilize the mRNA and/or repress translation of the targeted mRNA. miRNAs are expressed in the nervous system and modulate large numbers of neuronal genes, playing important roles in neurogenesis and neurodegenerative diseases. Besides being a major level of gene expression regulation, miRNAs also are recognized as excellent biomarkers for various diseases, especially in the diagnosis and prognosis of cancer. However, the roles of miRNAs in the pathogenesis of Parkinson's Disease and their potential as biomarkers for Parkinson's Disease have not being fully studied.

SUMMARY

In some aspects, the disclosure relates to pharmaceutical compositions including an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be a) complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283, or b) hybridizes under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1-283.

In further aspects, the disclosure relates to methods of diagnosing Parkinson's Disease (PD) in a subject. The methods may include detecting the level of expression of at least one miRNA of SEQ ID NOs: 1-283 in a biological sample from the subject, and comparing the level of expression in the sample to the level of expression in a reference. An increased or decreased level of expression in the sample compared to the level of expression in the reference may identify the subject as having PD or who is at risk of developing PD. In some aspects, a miRNA signature may be used to diagnose a subject at risk for developing or having PD.

Another aspect of the disclosure provides methods for treating, preventing, or reducing the risk of PD associated with aberrant expression of a miRNA in a cell, tissue, or animal, the method comprising contacting the cell, tissue, or animal with a pharmaceutical composition including an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be a) complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283, or b) hybridizes under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1-283.

In yet another aspect of the disclosure, kits are provided for performing measurement of a miRNA signature, the miRNA signature comprising at least one miRNA of SEQ ID NOs: 1-283, wherein the kit comprises reagents for measuring an expression level of at least one miRNA of SEQ ID NOs: 1-283.

The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a heat-map of miRNAs differentially expressed in the putamen by clinical diagnosis.

FIG. 2 is a heat-map of miRNAs differentially expressed in the putamen by unified staging system for Lewy-body disorders (LBDs).

FIG. 3 is a heat-map of miRNAs differentially expressed in cerebrospinal fluid (CSF) by clinical diagnosis.

FIG. 4 is a heat-map of miRNAs differentially expressed in CSF by unified staging system of LBDs.

FIG. 5 is a diagram of miRNA signatures that may be used to reflect the onset of Lewy body (LB) pathology.

FIG. 6 is a diagram of miRNA signatures that may be used to reflect the onset of LB pathology.

FIG. 7 is a diagram of miRNA signatures that may be used to reflect the onset of PD.

FIG. 8 is a diagram of miRNA signatures that may be used to reflect the onset of PD.

FIG. 9 is a diagram of miRNA signatures that may be used to reflect the onset of cognitive impairment in PD.

FIG. 10 is a diagram of the genomic organization of miR-212/132 in the first intron of a non-coding RNA gene on human Chr1p13.3.

FIGS. 11A-D are graphs of the expression of miR-132 and miR-212.

DETAILED DESCRIPTION

In a broad sense, the disclosure relates to therapeutic and diagnostic markers for Parkinson's Disease (PD). The present invention relates to the discovery of differential expression levels of various miRNAs in PD compared to normal tissue.

miRNAs are newly recognized, small, regulatory RNAs, which regulate gene expression by repressing translation and/or break down mRNAs of their downstream target genes. Misregulation of miRNAs in the central nervous system contributes to neurodegenerative disorders. To identify miRNAs involved in the pathogenesis of PD, fresh frozen putamen and cerebrospinal fluid (CSF) samples of 10 normal control, 12 (ILBD), 10 PD, and 10 PD with dementia but no Alzheimer's disease (AD) (PDDnoAD) cases were obtained from the Sun Health Research Institute (SHRI) (Table 1 and Table 2). By Unified Staging System for Lewy body diseases (LBDs), besides 9 normal controls, these samples include 12 Stage 2, 9 Stage 3, and 11 Stage 4 samples, representing increasing severity of the diseases.

TABLE 1 Subjects Classified by Clinical Diagnosis and Unified Staging System for LBDs (USSLBD) Stages Diagnosis Stage 0 Stage 2 Stage 3 Stage 4 total Control 9 9 ILBD 7 5 12 PD 3 2 5 10 PDD no AD 2 2 6 10 Total 9 12 9 11 41 Beach T G et al. Unified staging system for Lewy body disorders (LBDs): correlation with nigrostriatal degeneration, cognitive impairment and motor dysfunction. Acta Neuropathol. 2009, 117; 613-634. Classification of subjects with LBDs by Lewy-type α-synucleinopathy (LTS) distributions: Stage 0: No LTS. Control group. Stage 1: Olfactory bulb only; Stage 2: Brainstem or Limbic predominant; Stage 3: Brainstem and Limbic; Stage 4: Neocortical.

TABLE 2 Main basic characteristics of the study subjects classified by clinical diagnosis. SN pigmented α-Synuclein gender ApoE-s4 PMI neuron density score No. age* (% M) (%) (mean +/− SD) MMSE** UPDRS*** loss score**** (average +/− SD) control 9 82.1 +/− 6.8 60 22.0 2.4 +/− 0.6 29.0 (1.3) 7.0 (5.4) none (6), mild (3) 0 (73-97) (9) (9) ILBD 12  86.3 +/− 4.85 67 16.7 2.7 +/− 0.6 28.0 (1.7) 7.0 (5.1) none (5), mild (3),  8.6 +/− 8.1 (73-91) (8) (7) mod (1), severe (3) PD 10 79.4 +/− 5.9 55 18.2 6.2 +/− 4.2 26.8 (3.3)  9.6 (14.9) mile (1), mod (2), 18.0 +/− 5.3 (72-90) (4) (6) severe (8) PDD 10 76.1 +/− 5.9 60 20.0 3.0 +/− 1.4  17.1 (10.0) 25.3 (39.3) none (1), severe (9) 19.1 +/− 5.2 (no AD) (69-84) (7) (6) *age is presented by average +/− standard deviation (range); **MMSE is presented by, average (standard deviation) (number of objects with MMSE data); ***UPDRS is presented by average (standard deviation) (number of objects with MMSE data), and was obtained from subjects who were in the “off state” with respect to dopaminergic therapeutic effects.

Subsequently, miRNA-proof total RNA from all putamen and CSF samples was isolated using a mirVana™ miRNA isolation system (Life Technologies). On average, 0.69+/−0.06 μg of total RNA/mg putamen tissue (n=41); and 2.74+/−0.21 μg of total RNA/mL CSF samples (n=41) was obtained. There was no significant difference in quantity of total RNA isolated from different diagnostic groups. Subsequently, miRNA profiling in all putamen (n=41) and CSF total RNA (n=41) was performed using the Taqman miRNA microarrays (Applied Biosystem), which detected 754 known human miRNAs in total, to establish miRNA transcriptomes of the putamen and CSF of all 41 cases.

By comparison of these miRNA-expression profiles, miRNAs differentially expressed in the putamen and CSF among different groups classified by clinical diagnosis, by Unified Staging System for LBD, and by the extent of Substantia Nigra (SN) neuron depigmentation were identifed. As further detailed in the Examples, expression profiles were analyzed using a two-tailed t test between two groups. Significant differential expression was identified as p<0.05 and ≧2 fold changes, including both increased and decreased expression levels by ≧2 folds. Exemplary miRNAs (SEQ ID NOs: 1-283) are shown in Table 39. In some embodiments, at least one miRNA selected from SEQ ID NOs: 1-283 may have increased or decreased expression relative to a reference, control, or normal sample by at least about 1.5-fold, at least about 1.6-fold, at least about 1.7-fold, at least about 1.8-fold, at least about 1.9-fold, at least about 2.0-fold, at least about 2.1-fold, at least about 2.2-fold, at least about 2.3-fold, at least about 2.4-fold, at least about 2.5-fold, at least about 2.6-fold, at least about 2.7-fold, at least about 2.8-fold, at least about 2.9-fold, at least about 3.0-fold, at least about 3.1-fold, at least about 3.2-fold, at least about 3.3-fold, at least about 3.4-fold, at least about 3.5-fold, at least about 3.6-fold, at least about 3.7-fold, at least about 3.8-fold, at least about 3.9-fold, or at least about 4.0-fold.

TABLE 39 Sequences of exemplary miRNAs. SEQ miRNA Sequences ID NO miRNA signature hsa_miR_590_3P_002677 UAAUUUUAUGUAUAAGCUAGU 1 distinguishing hsa_miR_213_000516 ACCAUCGACCGUUGAUUGUACC 2 control vs PD hsa_miR_9#_002231 AUAAAGCUAGAUAACCGAAAGU 3 hsa_miR_191#_002678 GCUGCGCUUGGAUUUCGUCCCC 4 hsa_miR_497_001043 CAGCAGCACACUGUGGUUUGU 5 hsa_miR_664_002897 UAUUCAUUUAUCCCCAGCCUACA 6 hsa_miR_99a#_002141 CAAGCUCGCUUCUAUGGGUCUG 7 hsa_miR_1183_002841 CACUGUAGGUGAUGGUGAGAGUGGGCA 8 hsa_miR_340#_002259 UCCGUCUCAGUUACUUUAUAGC 9 hsa_miR_628_3p_002434 UCUAGUAAGAGUGGCAGUCGA 10 rno_miR_7#_001338 CAACAAAUCACAGUCUGCCAUA 11 hsa_miR_29a#_002447 ACUGAUUUCUUUUGGUGUUCAG 12 hsa_miR_142_3p_4373136 UGUAGUGUUUCCUACUUUAUGGA 13 miRNA signature hsa-miR-590-3P-002677 UAAUUUUAUGUAUAAGCUAGU 14 distinguishing hsa-miR-664-002897 UAUUCAUUUAUCCCCAGCCUACA 15 (Control + ILBD) hsa-miR-519a-4395526 AAAGUGCAUCCUUUUAGAGUGU 16 vs PD hsa-miR-340#-002259 UCCGUCUCAGUUACUUUAUAGC 17 hsa-miR-720-002895 UCUCGCUGGGGCCUCCA 18 hsa-miR-142-3p-4373136 UGUAGUGUUUCCUACUUUAUGGA 19 hsa-miR-185-4395382 UGGAGAGAAAGGCAGUUCCUGA 20 hsa-miR-213-000516 ACCAUCGACCGUUGAUUGUACC 21 miRNA signature hsa-miR-664-002897 UAUUCAUUUAUCCCCAGCCUACA 22 distinguishing ILBD hsa-miR-1285-002822 UCUGGGCAACAAAGUGAGACCU 23 vs PD hsa-miR-1183-002841 CACUGUAGGUGAUGGUGAGAGUGGGCA 24 hsa-miR-143-4395360 UGAGAUGAAGCACUGUAGCUCA 25 hsa-miR-519a-4395526 AAAGUGCAUCCUUUUAGAGUGU 26 hsa-miR-603-001566 CACACACUGCAAUUACUUUUGC 27 miRNA signature hsa_miR_590_3P_002677 UAAUUUUAUGUAUAAGCUAGU 28 distinguishing PD hsa_miR_213_000516 ACCAUCGACCGUUGAUUGUACC 29 vs PDDnoAD hsa_miR_409_3p_002332 GAAUGUUGCUCGGUGAACCCCU 30 hsa_miR_500_4395539 UAAUCCUUGCUACCUGGGUGAGA 31 dme_miR_7_000268 UGGAAGACUAGUGAUUUUGUUGU 32 hsa_miR_206_000510 UGGAAUGUAAGGAAGUGUGUGG 33 hsa_miR_629_001562 GUUCUCCCAACGUAAGCCCAGC 34 All miRNA dme-miR-7-000268 UGGAAGACUAGUGAUUUUGUUGU 35 differentially hsa-let-7c-4373167 UGAGGUAGUAGGUUGUAUGGUU 36 expressed and/or hsa-let-7d-4395394 AGAGGUAGUAGGUUGCAUAGU 37 correlated with hsa-let-7f-1#-002417 CUAUACAAUCUAUUGCCUUCCC 38 clinical/pathological hsa-miR-100#-002142 CAAGCUUGUAUCUAUAGGUAUG 39 findings hsa-miR-101#-002143 UACAGUACUGUGAUAACUGAA 40 hsa-miR-101-4395364 CAGUUAUCACAGUGCUGAUGCU 41 hsa-miR-105-4395278 UCAAAUGCUCAGACUCCUGUGGU 42 hsa-miR-106b#-002380 CCGCACUGUGGGUACUUGCUGC 43 hsa-miR-107-4373154 AGCAGCAUUGUACAGGGCUAUCA 44 hsa-miR-10b-4395329 UACCCUGUAGAACCGAAUUUGUG 45 hsa-miR-1183-002841 CACUGUAGGUGAUGGUGAGAGUGGGCA 46 hsa-miR-1201-002781 AGCCUGAUUAAACACAUGCUCUGA 47 hsa-miR-122-4395356 UGGAGUGUGACAAUGGUGUUUG 48 hsa-miR-1224-3P-002752 CCCCACCUCCUCUCUCCUCAG 49 hsa-miR-1226#-002758 GUGAGGGCAUGCAGGCCUGGAUGGGG 50 hsa-miR-1227-002769 CGUGCCACCCUUUUCCCCAG 51 hsa-miR-1233-002768 UGAGCCCUGUCCUCCCGCAG 52 hsa-miR-1238-002927 CUUCCUCGUCUGUCUGCCCC 53 hsa-miR-1244-002791 AAGUAGUUGGUUUGUAUGAGAUGGUU 54 hsa-miR-124-4373295 UUAAGGCACGCGGUGAAUGCCA 55 hsa-miR-1248-002870 ACCUUCUUGUAUAAGCACUGUGCUAAA 56 hsa-miR-1255B-002801 CGGAUGAGCAAAGAAAGUGGUU 57 hsa-miR-1256-002850 AGGCAUUGACUUCUCACUAGCU 58 hsa-miR-125a-5p-4395309 UCCCUGAGACCCUUUAACCUGUGA 59 hsa-miR-125b2#-002158 UCACAAGUCAGGCUCUUGGGAC 60 hsa-miR-125b-4373148 UCCCUGAGACCCUAACUUGUGA 61 hsa-miR-126#-000451 CAUUAUUACUUUUGGUACGCG 62 hsa-miR-1260-002896 AUCCCACCUCUGCCACCA 63 hsa-miR-1264-002799 CAAGUCUUAUUUGAGCACCUGUU 64 hsa-miR-1269-002789 CUGGACUGAGCCGUGCUACUGG 65 hsa-miR-1270-002807 CUGGAGAUAUGGAAGAGCUGUGU 66 hsa-miR-1271-002779 CUUGGCACCUAGCAAGCACUCA 67 hsa-miR-127-5p-4395340 CUGAAGCUCAGAGGGCUCUGAU 68 hsa-miR-1282-002803 UCGUUUGCCUUUUUCUGCUU 69 hsa-miR-1290-002863 UGGAUUUUUGGAUCAGGGA 70 hsa-miR-1291-002838 UGGCCCUGACUGAAGACCAGCAGU 71 hsa-miR-129-3p-4373297 AAGCCCUUACCCCAAAAAGUAU 72 hsa-miR-1298-002861 UUCAUUCGGCUGUCCAGAUGUA 73 hsa-miR-1300-002902 UUGAGAAGGAGGCUGCUG 74 hsa-miR-1301-002827 UUGCAGCUGCCUGGGAGUGACUUC 75 hsa-miR-1303-002792 UUUAGAGACGGGGUCUUGCUCU 76 hsa-miR-130a-4373145 CAGUGCAAUGUUAAAAGGGCAU 77 hsa-miR-130b-4373144 CAGUGCAAUGAUGAAAGGGCAU 78 hsa-miR-132-4373143 UAACAGUCUACAGCCAUGGUCG 79 hsa-miR-135a-4373140 UAUGGCUUUUUAUUCCUAUGUGA 80 hsa-miR-135b#-002159 AUGUAGGGCUAAAAGCCAUGGG 81 hsa-miR-135b-4395372 UAUGGCUUUUCAUUCCUAUGUGA 82 hsa-miR-136#-002100 CAUCAUCGUCUCAAAUGAGUCU 83 hsa-miR-137-4373301 UAUUGCUUAAGAAUACGCGUAG 84 hsa-miR-138-2#-002144 AGCUGGUGUUGUGAAUCAGGCCG 85 hsa-miR-138-4395395 GCUAUUUCACGACACCAGGGUU 86 hsa-miR-141-4373137 UAACACUGUCUGGUAAAGAUGG 87 hsa-miR-142-3p-4373136 UGUAGUGUUUCCUACUUUAUGGA 88 hsa-miR-143-4395360 UGAGAUGAAGCACUGUAGCUC 89 hsa-miR-14395333 UGGAAUGUAAAGAAGUAUGUA 90 hsa-miR-144#-002148 GGAUAUCAUCAUAUACUGUAAG 91 hsa-miR-144-002676 UACAGUAUAGAUGAUGUACU 92 hsa-miR-145#-002149 GGAUUCCUGGAAAUACUGUUCU 93 hsa-miR-145-4395389 GUCCAGUUUUCCCAGGAAUCCCU 94 hsa-miR-146a#-002163 CCUCUGAAAUUCAGUUCUUCAG 95 hsa-miR-146b-5p-4373178 UGAGAACUGAAUUCCAUAGGCU 96 hsa-miR-148a-4373130 UCAGUGCACUACAGAACUUUGU 97 hsa-miR-148b#-002160 AAGUUCUGUUAUACACUCAGGC 98 hsa-miR-149#-002164 AGGGAGGGACGGGGGCUGUGC 99 hsa-miR-151-5P-002642 UCGAGGAGCUCACAGUCUAGU 100 hsa-miR-153-4373305 UUGCAUAGUCACAAAAGUGA 101 hsa-miR-154#-000478 AAUCAUACACGGUUGACCUAUU 102 hsa-miR-15a-4373123 UAGCAGCACAUAAUGGUUUGUG 103 hsa-miR-181a2#-002317 ACCACUGACCGUUGACUGUACC 104 hsa-miR-181a-4373117 AACAUUCAACGCUGUCGGUGAGU 105 hsa-miR-183#-002270 UAUGGCACUGGUAGAAUUCACU 106 hsa-miR-183-4395380 GUGAAUUACCGAAGGGCCAUAA 107 hsa-miR-184-4373113 UGGACGGAGAACUGAUAAGGGU 108 hsa-miR-18a-4395533 UAAGGUGCAUCUAGUGCAGAUAG 109 hsa-miR-191#-002678 GCUGCGCUUGGAUUUCGUCCCC 110 hsa-miR-192-4373108 CUGACCUAUGAAUUGACAGCC 111 hsa-miR-193a-5p-4395392 UGGGUCUUUGCGGGCGAGAUGA 112 hsa-miR-195-4373105 UAGCAGCACAGAAAUAUUGGC 113 hsa-miR-196b-4395326 UAGGUAGUUUCCUGUUGUUGGG 114 hsa-miR-197-4373102 UUCACCACCUUCUCCACCCAGC 115 hsa-miR-198-4395384 GGUCCAGAGGGGAGAUAGGUUC 116 hsa-miR-199a-5p-4373272 CCCAGUGUUCAGACUACCUGUUC 117 hsa-miR-19b-4373098 UGUGCAAAUCCAUGCAAAACUGA 118 hsa-miR-200b-4395362 UAAUACUGCCUGGUAAUGAUGA 119 hsa-miR-202-4395474 AGAGGUAUAGGGCAUGGGAA 120 hsa-miR-203-4373095 GUGAAAUGUUUAGGACCACUAG 121 hsa-miR-206-000510 UGGAAUGUAAGGAAGUGUGUGG 122 hsa-miR-211-4373088 UUCCCUUUGUCAUCCUUCGCCU 123 hsa-miR-212-4373087 UAACAGUCUCCAGUCACGGCC 124 hsa-miR-213-000516 ACCAUCGACCGUUGAUUGUACC 125 hsa-miR-21-4373090 UAGCUUAUCAGACUGAUGUUGA 126 hsa-miR-217-4395448 UACUGCAUCAGGAACUGAUUGGA 127 hsa-miR-218-4373081 UUGUGCUUGAUCUAACCAUGU 128 hsa-miR-219-2-3p-4395501 AGAAUUGUGGCUGGACAUCUGU 129 hsa-miR-22#-002301 AGUUCUUCAGUGGCAAGCUUUA 130 hsa-miR-220b-4395317 CCACCACCGUGUCUGACACUU 131 hsa-miR-222#-002097 CUCAGUAGCCAGUGUAGAUCCU 132 hsa-miR-223-4395406 UGUCAGUUUGUCAAAUACCCCA 133 hsa-miR-23a-4373074 AUCACAUUGCCAGGGAUUUCC 134 hsa-miR-24-1#-002440 UGCCUACUGAGCUGAUAUCAGU 135 hsa-miR-25-4373071 CAUUGCACUUGUCUCGGUCUGA 136 hsa-miR-26a-2#-002115 CCUAUUCUUGAUUACUUGUUUC 137 hsa-miR-26b#-002444 CCUGUUCUCCAUUACUUGGCUC 138 hsa-miR-27a#-002445 AGGGCUUAGCUGCUUGUGAGCA 139 hsa-miR-27b-4373068 UUCACAGUGGCUAAGUUCUGC 140 hsa-miR-28-3p-4395557 CACUAGAUUGUGAGCUCCUGGA 141 hsa-miR-296-3p-4395212 GAGGGUUGGGUGGAGGCUCUCC 142 hsa-miR-296-5p-4373066 AGGGCCCCCCCUCAAUCCUGU 143 hsa-miR-29a#-002447 ACUGAUUUCUUUUGGUGUUCAG 144 hsa-miR-29a-4395223 UAGCACCAUCUGAAAUCGGUUA 145 hsa-miR-29c-4395171 UAGCACCAUUUGAAAUCGGUUA 146 hsa-miR-302a-4378070 UAAGUGCUUCCAUGUUUUGGUGA 147 hsa-miR-302d-000535 UAAGUGCUUCCAUGUUUGAGUGU 148 hsa-miR-30a-3p-000416 CUUUCAGUCGGAUGUUUGCAGC 149 hsa-miR-30b-4373290 UGUAAACAUCCUACACUCAGCU 150 hsa-miR-30c-1#-002108 CUGGGAGAGGGUUGUUUACUCC 151 hsa-miR-30e-3p-000422 CUUUCAGUCGGAUGUUUACAGC 152 hsa-miR-31#-002113 UGCUAUGCCAACAUAUUGCCAU 153 hsa-miR-31-4395390 AGGCAAGAUGCUGGCAUAGCU 154 hsa-miR-320B-002844 AAAAGCUGGGUUGAGAGGGCAA 155 hsa-miR-324-5p-4373052 CGCAUCCCCUAGGGCAUUGGUGU 156 hsa-miR-326-4373050 CCUCUGGGCCCUUCCUCCAG 157 hsa-miR-328-4373049 CUGGCCCUCUCUGCCCUUCCGU 158 hsa-miR-330-5p-4395341 UCUCUGGGCCUGUGUCUUAGGC 159 hsa-miR-337-3p-002157 CUCCUAUAUGAUGCCUUUCUUC 160 hsa-miR-338-3p-4395363 UCCAGCAUCAGUGAUUUUGUUG 161 hsa-miR-339-5p-4395368 UCCCUGUCCUCCAGGAGCUCACG 162 hsa-miR-33b-4395196 GUGCAUUGCUGUUGCAUUGC 163 hsa-miR-340#-002259 UCCGUCUCAGUUACUUUAUAGC 164 hsa-miR-340-4395369 UUAUAAAGCAAUGAGACUGAUU 165 hsa-miR-345-4395297 GCUGACUCCUAGUCCAGGGCUC 166 hsa-miR-34a#-002316 CAAUCAGCAAGUAUACUGCCCU 167 hsa-miR-34a-4395168 UGGCAGUGUCUUAGCUGGUUGU 168 hsa-miR-34b-000427 UAGGCAGUGUCAUUAGCUGAUUG 169 hsa-miR-34c-5p-4373036 AGGCAGUGUAGUUAGCUGAUUGC 170 hsa-miR-362-5p-4378092 AAUCCUUGGAACCUAGGUGUGAGU 171 hsa-miR-363#-001283 CGGGUGGAUCACGAUGCAAUUU 172 hsa-miR-363-4378090 AAUUGCACGGUAUCCAUCUGUA 173 hsa-miR-365-4373194 UAAUGCCCCUAAAAAUCCUUAU 174 hsa-miR-367-4373034 AAUUGCACUUUAGCAAUGGUGA 175 hsa-miR-373-4378073 GAAGUGCUUCGAUUUUGGGGUGU 176 hsa-miR-374b#-002391 CUUAGCAGGUUGUAUUAUCAUU 177 hsa-miR-374b-4381045 AUAUAAUACAACCUGCUAAGUG 178 hsa-miR-376c-4395233 AACAUAGAGGAAAUUCCACGU 179 hsa-miR-378-000567 CUCCUGACUCCAGGUCCUGUGU 180 hsa-miR-380-5p-000570 UGGUUGACCAUAGAACAUGCGC 181 hsa-miR-381-4373020 UAUACAAGGGCAAGCUCUCUGU 182 hsa-miR-382-4373019 GAAGUUGUUCGUGGUGGAUUCG 183 hsa-miR-383-4373018 AGAUCAGAAGGUGAUUGUGGCU 184 hsa-miR-409-3p-002332 GAAUGUUGCUCGGUGAACCCCU 185 hsa-miR-411-4381013 UAGUAGACCGUAUAGCGUACG 186 hsa-miR-424#-002309 CAAAACGUGAGGCGCUGCUAU 187 hsa-miR-424-4373201 CAGCAGCAAUUCAUGUUUUGAA 188 hsa-miR-431-4395173 UGUCUUGCAGGCCGUCAUGCA 189 hsa-miR-432-001026 UCUUGGAGUAGGUCAUUGGGUGG 190 hsa-miR-448-4373206 UUGCAUAUGUAGGAUGUCCCAU 191 hsa-miR-449b-4381011 AGGCAGUGUAUUGUUAGCUGGC 192 hsa-miR-450a-4395414 UUUUGCGAUGUGUUCCUAAUAU 193 hsa-miR-454-4395434 UAGUGCAAUAUUGCUUAUAGGGU 194 hsa-miR-483-3p-002339 UCACUCCUCUCCUCCCGUCUU 195 hsa-miR-484-4381032 UCAGGCUCAGUCCCCUCCCGAU 196 hsa-miR-486-5p-4378096 UCCUGUACUGAGCUGCCCCGAG 197 hsa-miR-488-4395468 UUGAAAGGCUAUUUCUUGGUC 198 hsa-miR-489-4395469 GUGACAUCACAUAUACGGCAGC 199 hsa-miR-497-001043 CAGCAGCACACUGUGGUUUGU 200 hsa-miR-499-5p-4381047 UUAAGACUUGCAGUGAUGUUUAA 201 hsa-miR-500-001046 AUGCACCUGGGCAAGGAUUCUG 202 hsa-miR-511-4373236 GUGUCUUUUGCUCUGCAGUCA 203 hsa-miR-516-3p-001149 UGCUUCCUUUCAGAGGGU 204 hsa-miR-516b-4395172 AUCUGGAGGUAAGAAGCACUUU 205 hsa-miR-517#-001113 CCUCUAGAUGGAAGCACUGUCU 206 hsa-miR-518a-3p-4395508 GAAAGCGCUUCCCUUUGCUGGA 207 hsa-miR-518b-4373246 CAAAGCGCUCCCCUUUAGAGGU 208 hsa-miR-519a-4395526 AAAGUGCAUCCUUUUAGAGUGU 209 hsa-miR-520c-3p-002400 AAAGUGCUUCCUUUUAGAGGGU 210 hsa-miR-520D-3P-002743 AAAGUGCUUCUCUUUGGUGGGU 211 hsa-miR-520g-4373257 ACAAAGUGCUUCCCUUUAGAGUGU 212 hsa-miR-521-4373259 AACGCACUUCCCUUUAGAGUGU 213 hsa-miR-539-4378103 GGAGAAAUUAUCCUUGGUGUGU 214 hsa-miR-541-4395312 UGGUGGGCACAGAAUCUGGACU 215 hsa-miR-543-002376 AAACAUUCGCGGUGCACUUCUU 216 hsa-miR-545-4395378 UCAGCAAACAUUUAUUGUGUGC 217 hsa-miR-548b-5p-4395519 AAAAGUAAUUGUGGUUUUGGCC 218 hsa-miR-548c-5p-4395540 AAAAGUAAUUGCGGUUUUUGCC 219 hsa-miR-5481-002909 AAAAGUAAUUGCGGAUUUUGCC 220 hsa-miR-551b#-002346 GAAAUCAAGCGUGGGUGAGACC 221 hsa-miR-552-001520 AACAGGUGACUGGUUAGACAA 222 hsa-miR-559-001527 UAAAGUAAAUAUGCACCAAAA 223 hsa-miR-566-001533 GGGCGCCUGUGAUCCCAAC 224 hsa-miR-571-001613 UGAGUUGGCCAUCUGAGUGAG 225 hsa-miR-574-3p-4395460 CACGCUCAUGCACACACCCACA 226 hsa-miR-577-002675 UAGAUAAAAUAUUGGUACCUG 227 hsa-miR-582-3p-4395510 UAACUGGUUGAACAACUGAACC 228 hsa-miR-589-001543 UCAGAACAAAUGCCGGUUCCCAGA 229 hsa-miR-590-3P-002677 UAAUUUUAUGUAUAAGCUAGU 230 hsa-miR-590-5p-4395176 GAGCUUAUUCAUAAAAGUGCAG 231 hsa-miR-592-001546 UUGUGUCAAUAUGCGAUGAUGU 232 hsa-miR-593-001547 AGGCACCAGCCAGGCAUUGCUCAGC 233 hsa-miR-597-4380960 UGUGUCACUCGAUGACCACUGU 234 hsa-miR-601-001558 UGGUCUAGGAUUGUUGGAGGAG 235 hsa-miR-604-001567 AGGCUGCGGAAUUCAGGAC 236 hsa-miR-608-001571 AGGGGUGGUGUUGGGACAGCUCCGU 237 hsa-miR-616-4395525 AGUCAUUGGAGGGUUUGAGCAG 238 hsa-miR-617-001591 AGACUUCCCAUUUGAAGGUGGC 239 hsa-miR-618-4380996 AAACUCUACUUGUCCUUCUGAGU 240 hsa-miR-622-001553 ACAGUCUGCUGAGGUUGGAGC 241 hsa-miR-625#-002432 GACUAUAGAACUUUCCCCCUCA 242 hsa-miR-626-001559 AGCUGUCUGAAAAUGUCUU 243 hsa-miR-628-3p-002434 UCUAGUAAGAGUGGCAGUCGA 244 hsa-miR-628-5p-4395544 AUGCUGACAUAUUUACUAGAGG 245 hsa-miR-629-001562 UGGGUUUACGUUGGGAGAACU 246 hsa-miR-630-001563 AGUAUUCUGUACCAGGGAAGGU 247 hsa-miR-635-001578 ACUUGGGCACUGAAACAAUGUCC 248 hsa-miR-638-001582 AGGGAUCGCGGGCGGGUGGCGGCCU 249 hsa-miR-639-001583 AUCGCUGCGGUUGCGAGCGCUGU 250 hsa-miR-641-001585 AAAGACAUAGGAUAGAGUCACCUC 251 hsa-miR-644-001596 AGUGUGGCUUUCUUAGAGC 252 hsa-miR-649-001602 AAACCUGUGUUGUUCAAGAGUC 253 hsa-miR-652-4395463 AAUGGCGCCACUAGGGUUGUG 254 hsa-miR-654-3p-4395350 UAUGUCUGCUGACCAUCACCUU 255 hsa-miR-659-001514 CUUGGUUCAGGGAGGGUCCCCA 256 hsa-miR-660-4380925 UACCCAUUGCAUAUCGGAGUUG 257 hsa-miR-661-001606 UGCCUGGGUCUCUGGCCUGCGCGU 258 hsa-miR-663B-002857 GGUGGCCCGGCCGUGCCUGAGG 259 hsa-miR-664-002897 UAUUCAUUUAUCCCCAGCCUACA 260 hsa-miR-671-3p-4395433 UCCGGUUCUCAGGGCUCCACC 261 hsa-miR-708-4395452 AAGGAGCUUACAAUCUAGCUGGG 262 hsa-miR-720-002895 UCUCGCUGGGGCCUCCA 263 hsa-miR-758-4395180 UUUGUGACCUGGUCCACUAACC 264 hsa-miR-767-5p-001993 UGCACCAUGGUUGUCUGAGCAUG 265 hsa-miR-769-5p-001998 UGAGACCUCUGGGUUCUGAGCU 266 hsa-miR-873-4395467 GCAGGAACUUGUGAGUCUCCU 267 hsa-miR-876-3p-4395336 UGGUGGUUUACAAAGUAAUUCA 268 hsa-miR-885-5p-4395407 UCCAUUACACUACCCUGCCUCU 269 hsa-miR-886-3p-4395305 CGCGGGUGCUUACUGACCCUU 270 hsa-miR-886-5p-4395304 CGGGUCGGAGUUAGCUCAAGCGG 271 hsa-miR-9#-002231 AUAAAGCUAGAUAACCGAAAGU 272 hsa-miR-920-002150 GGGGAGCUGUGGAAGCAGUA 273 hsa-miR-922-002152 GCAGCAGAGAAUAGGACUACGUC 274 hsa-miR-92a-1#-002137 AGGUUGGGAUCGGUUGCAAUGCU 275 hsa-miR-92a-4395169 UAUUGCACUUGUCCCGGCCUGU 276 hsa-miR-9-4373285 UCUUUGGUUAUCUAGCUGUAUGA 277 hsa-miR-98-4373009 UGAGGUAGUAAGUUGUAUUGUU 278 hsa-miR-99a#-002141 CAAGCUCGCUUCUAUGGGUCUG 279 hsa-miR-99b-4373007 CACCCGUAGAACCGACCUUGCG 280 mmu-let-7d#-001178 CUAUACGACCUGCUGCCUUUCU 281 rno-miR-29c#-001818 UGACCGAUUUCUCCUGGUGUUC 282 rno-miR-7#-001338 CAACAAAUCACAGUCUGCCAUA 283

As shown in the Examples discovered are miRNAs that are useful as diagnostic markers and therapeutic targets for the treatment and prevention of Parkinson's Disease and related disorders. As used herein, disorders related to Parkinson's Disease may include, but are not limited to, Alzheimer's, dementia, cognitive impairment, pre-symptomatic Parkinson's Disease, and Lewis Body disease.

miRNA or microRNA refer to 19-25 nucleotide (nt) non-coding RNAs derived from endogenous genes that act as post-transcriptional regulators of gene expression. They are processed from longer (ca 70-80 nt) hairpin-like precursors termed pre-miRNAs by the RNAse III enzyme Dicer. miRNAs assemble in ribonucleoprotein complexes termed miRNPs and recognise their target sites by antisense complementarity thereby mediating down-regulation of their target genes. Near-perfect or perfect complementarity between the miRNA and its target site results in target mRNA cleavage, whereas limited complementarity between the miRNA and the target site results in translational inhibition of the target gene.

Compositions

In certain aspects, provided are pharmaceutical compositions. The pharmaceutical compositions may include an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283. The oligonucleotide may be complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283. The oligonucleotide may hybridize under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1-283. The oligonucleotide may comprise any nucleic acid. “Nucleic acid” includes double- and single-stranded DNA of different lengths, as well as double- and single stranded RNA of different lengths, or synthetic variants thereof. Thus, the nucleic acid may be, for example, DNA, cDNA, RNA, sRNA, locked nucleotide acid (LNA), HNA, peptide nucleic acid (PNA), or any other variant hereof.

As used herein, “complementary” or “substantially complementary” refers to the hybridization or base pairing between nucleotides or nucleic acids, such as, for instance, between the two strands of a double stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single stranded nucleic acid to be sequenced or amplified. Complementary nucleotides are, generally, A and T (or A and U), or C and G. Two single stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and with appropriate nucleotide insertions or deletions, pair with at least about 80% of the nucleotides of the other strand, usually at least about 90% to 95%, and more preferably from about 98 to 100%

The oligonucleotide may be at least about 10 nucleotides (nt), at least about 11 nt, at least about 12 nt, at least about 13 nt, at least about 14 nt, at least about 15 nt, at least about 16 nt, at least about 17 nt, at least about 18 nt, at least about 19 nt, or at least about 20 nt in length. The oligonucleotide may be less than about 100 nt, less than about 90 nt, less than about 80 nt, less than about 70 nt, less than about 60 nt, or less than about 50 nt in length. This may include ranges of about 10 nt to about 50 nt, about 12 nt to about 40 nt, or about 15 nt to about 30 nt in length.

The oligonucleotide may be capable of reducing expression of the miRNA by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, or at least about 60%. The oligonucleotide may be capable of reducing expression of the miRNA by less than about 99%, less than about 98%, less than about 95%, less than about 90%, less than about 85%, less than about 80%, or less than about 75%. This may include ranges of about 10% to about 99%, about 20% to about 98%, about 30% to about 90%, or about 40% to about 80%.

The oligonucleotide may be chemically modified. Chemical modifications may include addition of an imaging agent or reporter molecule. As used herein, an “imaging agent” or “reporter molecule” is any entity which enhances visualization or detection of the oligonucleotide. Any type of detectable reporter molecule/imaging agent can be used in the methods disclosed herein. Such detectable molecules are known in the art and include, for example, magnetic beads, fluorophores, radionuclides, nuclear stains (e.g., DAPI). For example, an imaging agent can include a compound that comprises an unstable isotope (i.e., a radionuclide) or a fluorescent moiety, such as Cy-5, Alexa 647, Alexa 555, Alexa 488, fluorescein, rhodamine, and the like. Suitable radionuclides include both alpha- and beta-emitters. In some embodiments, the targeting vehicle is labeled. In other embodiments, suitable radioactive moieties include labeled polynucleotides and polypeptides which can be coupled to the targeting vehicle.

In some embodiments, the imaging agent comprises a radionuclide such as, for example, a radionuclide that emits low-energy electrons (e.g., those that emit photons with energies as low as 20 keV). Such nuclides can irradiate the cell to which they are delivered without irradiating surrounding cells or tissues. Non-limiting examples of radionuclides that are can be delivered to cells include ¹³⁷Cs, ¹⁰³Pd, ¹¹¹In, ¹²⁵I, ²¹¹At, ²¹²Bi and ²¹³Bi, among others known in the art. Further imaging agents suitable for delivery to a cell in accordance with some embodiments include paramagnetic species for use in MRI imaging, echogenic entities for use in ultrasound imaging, fluorescent entities for use in fluorescence imaging (including quantum dots), and light-active entities for use in optical imaging. A suitable species for MRI imaging is a gadolinium complex of diethylenetriamine pentacetic acid (DTPA). For positron emission tomography (PET), ¹⁸F or ¹¹C may be delivered.

Methods

In further aspects, the disclosure relates to methods of diagnosing Parkinson's Disease (PD) in a subject. The methods may include detecting the level of expression of at least one miRNA of SEQ ID NOs: 1-283 in a biological sample from the subject, and comparing the level of expression in the sample to the level of expression in a reference. An increased or decreased level of expression in the sample compared to the level of expression in the reference may identify the subject as having PD or who is at risk of developing PD.

Various detection methods that can be employed for the methods described herein, and the present invention includes all the mentioned methods, but is not limited to any of these. Detection methods may include use of a probe. A probe is a specific sequence of a nucleic acid used to detect nucleic acids by hybridization. A probe may be labelled, tagged or immobilized or otherwise modified according to the requirements of the detection method chosen. A label or a tag is an entity making it possible to identify a compound to which it is associated. It is within the scope of the present invention to employ probes that are labelled or tagged by any means known in the art such as but not limited to: radioactive labelling, fluorescent labelling and enzymatic labelling. Furthermore the probe, labeled or not, may be immobilized to facilitate detection according to the detection method of choice and this may be accomplished according to the preferred method of the particular detection method. Detection methods may also include in situ hybridization, PCR, microarrays, Northern blot analysis, and affinity matrices.

In some embodiments, RT-PCR may be used as the detection method. In some embodiments, the detection method may include quantitative real-time RT-PCR, for example, the TaqMan® MicroRNA assay (Applied Biosystems) may be used for detection of expression of the miRNAs described herein.

In some embodiments, a kit may be provided with reagents to measure at least one of the miRNAs. In some embodiments, a kit may be provided to measure a miRNA signature. By way of non-limiting example, the kit may include reagents to measure a miRNA signature where the miRNA signature includes at least one of SEQ ID NOs: 204, 110, 192, 200, 244, 21, 134, 144, 198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b, miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#) or a combination thereof and in some embodiments includes all 14. An embodiment of the kit may include reagents to measure a miRNA signature where the miRNA signature includes at least one of SEQ ID NOs: 1-13, (miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p) or combinations thereof and in some embodiments includes all 13. An embodiment of the kit may include reagents to measure a miRNA signature where the miRNA signature includes at least one of SEQ ID NOs: 14-21 (miR-590-3p, miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185, and miR-213) or combinations thereof an in some embodiments includes all 8. An embodiment of the kit may include reagents to measure a miRNA signature where the miRNA signature includes at least one of SEQ ID NOs: 22-27 (miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603) or combinations thereof an in some embodiments includes all 6. An embodiment of the kit may include reagents to measure a miRNA signature where the miRNA signature includes at least one of SEQ ID NOs: 28-34 (miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629) or combinations thereof an in some embodiments includes all 7. Additional combinations for the kit for measuring a miRNA signature are also possible.

In some embodiments, the expression level of one or more miRNAs of SEQ ID NOS 1-283 may be compared to a reference expression level using statistical analysis with a computer to implement the statistical analysis. In some embodiments Support Vector Machine Recursive Feature Selection (SVM-RFE) and SVM classification analysis from JMP Genomics (SAS Institute) is used to compare the miRNA expression levels. In some embodiments, the data for the array assays will be first analyzed using RQ manager 1.2 software (Applied Biosystems) to obtain raw expression levels of all the miRNAs in each sample, represented by C^(T). In some embodiments, the miRNA expression profiles of ILBD, PD, and PDDnoAD patients may be compared to age-matched normal controls in the CSF or SNs. The miRNA profiles of ILBD, PD, and PDDnoAD patients may be compared to each other. Changes in the levels of miRNA expression in each pair will be evaluated by the DDCt or Δ(ΔC^(T)). In some embodiments, StatMiner software (Integromics) may be used for bioinformatic and statistical analysis.

The subject may be any mammal. Mammals include including humans, mice, rats, rabbits, cats, and dogs. The subject is preferably human. The subject may be predisposed for a disease or suffering from a disease.

A sample may be a small part of a subject, representative of the whole, and may be constituted by a biopsy or a body fluid sample. Biopsies are small pieces of tissue and may be fresh, frozen or fixed, such as formalin-fixed and paraffin embedded (FFPE). Body fluid samples may be blood, plasma, serum, urine, sputum, cerebrospinal fluid, milk, or ductal fluid samples and may likewise be fresh, frozen or fixed. Suitably, the sample comprises blood. Samples may be removed surgically, by extraction, i.e. by hypodermic or other types of needles, by microdissection, or laser capture.

The reference may be any suitable control sample known in the art, such as, for example, a sample from a normal, healthy subject. The reference may be a sample from the same subject prior to demonstration of disease symptoms or prior to diagnosis with Parkinson's Disease. The reference may be a “standardized” sample, such as a sample comprising material or data from several samples, preferably also from several individuals. A standardized sample may comprise either normal or diseased sample material or data.

Before analyzing the sample, it may be desirable to perform one or more sample preparation operations upon the sample. Typically, these sample preparation operations will include such manipulations as concentration, suspension, extraction of intracellular material, e.g., nucleic acids from tissue/whole cell samples and the like, amplification of nucleic acids, fragmentation, transcription, labeling and/or extension reactions. Nucleic acids, especially RNA and specifically miRNA can be isolated using any techniques known in the art. There are two main methods for isolating RNA: phenol-based extraction and silica matrix or glass fiber filter (GFF)-based binding. Phenol-based reagents contain a combination of denaturants and RNase inhibitors for cell and tissue disruption and subsequent separation of RNA from contaminants. Phenol-based isolation procedures can recover RNA species in the 10-200-nucleotide range e.g., miRNAs, 5S rRNA, 5.8S rRNA, and U1 snRNA. If a sample of “total” RNA was purified by the popular silica matrix column or GFF procedure, it may be depleted in small RNAs. Extraction procedures such as those using Trizol or TriReagent, however will purify all RNAs, large and small, and are the recommended methods for isolating total RNA from biological samples that will contain miRNAs/siRNAs. Any method required for the processing of a sample prior to detection by any of the herein mentioned methods falls within the scope of the present invention. These methods are typically well known by a person skilled in the art.

Other aspects of the disclosure provide methods for treating, preventing, or reducing the risk of PD associated with aberrant expression of a miRNA in a cell, tissue, or animal. The methods may include contacting the cell, tissue, or animal with a pharmaceutical composition as detailed above. Aberrant expression includes up-regulation and down-regulation of the miRNA. In some embodiments, miRNA is up-regulated.

“Administration” or “administering,” as used herein, refers to providing, contacting, and/or delivery of a compound or compounds by any appropriate route to achieve the desired effect. Administration may include, but is not limited to, oral, sublingual, parenteral (e.g., intravenous, subcutaneous, intracutaneous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional or intracranial injection), transdermal, topical, buccal, rectal, vaginal, nasal, ophthalmic, via inhalation, and implants.

“Contacting,” as used herein as in “contacting a cell,” refers to contacting a cell directly or indirectly in vitro, ex vivo, or in vivo (i.e. within a subject, such as a mammal). Contacting a cell, which also includes “reacting” a cell, can occur as a result of administration to a subject. Contacting encompasses administration to a cell, tissue, mammal, subject, patient, or human. Further, contacting a cell includes adding an agent to a cell culture. Other suitable methods may include introducing or administering an agent to a cell, tissue, mammal, subject, or patient using appropriate procedures and routes of administration as defined herein.

As used herein, the term “treat” or “treating” a subject having a disorder refers to administering a regimen to the subject, such that at least one symptom of the disorder is cured, healed, alleviated, relieved, altered, remedied, ameliorated, or improved. Treating includes administering an amount effective to alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder or the symptoms of the disorder. The treatment may inhibit deterioration or worsening of a symptom of a disorder.

The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including but not limited to”) unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims.

EXAMPLES Example 1 Putamen miRNAs are Involved in the Pathogenesis of PD and are Novel Therapeutic Targets for Treatment of PD

Among the differentially expressed miRNAs in the putamen, we identified 51 miRNAs that are dysregulated among ILBD, PD and PDD, and normal controls (Tables 3-9; FIG. 1); 59 miRNAs that are dysregulated in Stages 2, 3, and 4 of Lewy Body diseases when compared to normal controls (Tables 10-16; FIG. 2).

TABLE 3 miRNAs Differentially Expressed in Putamen among Different Diagnostic Groups (p < 0.05; at least 2 fold change) (51 in total) Diagnosis Control ILBD PD PDDnoAD Control 27 (11, 16) 16 (7, 9) 11 (3, 8) ILBD 27 (11, 16) 14 (8, 6) 18 (10, 8) PD 16 (7, 9) 14 (8, 6) 12 (6, 6) PDDnoAD 11 (3, 8) 18 (10, 8) 12 (6, 6) *Numbers in parentheses are numbers of (upregulated, downregulated) miRNAs.

TABLE 4 miRNAs differentially expressed in ILBD compared to normal controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (ILBD/Ctl) hsa-miR-1224-3P-002752 0.007 10.69 hsa-miR-767-5p-001993 0.008 5.31 hsa-miR-431-4395173 0.009 4.25 mmu-let-7d#-001178 0.013 3.46 hsa-miR-183-4395380 0.000 3.37 hsa-miR-873-4395467 0.015 2.68 hsa-miR-33b-4395196 0.040 2.55 hsa-miR-516b-4395172 0.015 2.53 hsa-miR-127-5p-4395340 0.030 2.32 hsa-miR-489-4395469 0.001 2.08 hsa-miR-1270-002807 0.029 2.06 hsa-miR-34b-000427 0.008 0.20 hsa-miR-26a-2#-002115 0.002 0.22 hsa-miR-638-001582 0.000 0.22 hsa-miR-1290-002863 0.002 0.23 hsa-miR-302d-000535 0.016 0.23 hsa-miR-1248-002870 0.020 0.24 hsa-miR-922-002152 0.024 0.26 hsa-miR-30c-1#-002108 0.000 0.30 hsa-miR-92a-1#-002137 0.002 0.31 hsa-miR-497-001043 0.000 0.34 hsa-miR-1291-002838 0.005 0.36 hsa-miR-1244-002791 0.009 0.40 hsa-miR-635-001578 0.009 0.43 hsa-miR-601-001558 0.009 0.44 hsa-miR-1226#-002758 0.043 0.47 hsa-miR-106b#-002380 0.002 0.47

TABLE 5 miRNAs differentially expressed in PD compared to normal controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (PD/Ctl) hsa-miR-566-001533 0.01 15.96 hsa-miR-767-5p-001993 0.01 7.39 hsa-miR-1-4395333 0.01 2.94 hsa-miR-33b-4395196 0.03 2.88 hsa-miR-217-4395448 0.01 2.17 hsa-miR-424-4373201 0.04 2.17 hsa-miR-876-3p-4395336 0.02 2.12 hsa-miR-516-3p-001149 0.05 0.11 hsa-miR-644-001596 0.02 0.15 dme-miR-7-000268 0.05 0.27 hsa-miR-30c-1#-002108 0.01 0.38 hsa-miR-720-002895 0.04 0.39 hsa-miR-106b#-002380 0.00 0.42 hsa-miR-551b#-002346 0.02 0.46 hsa-miR-125b-2#-002158 0.02 0.48 hsa-miR-1256-002850 0.01 0.48

TABLE 6 miRNAs differentially expressed in PDD compared to normal controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (PDD/Ctl) hsa-miR-518a-3p-4395508 0.011 2.910 hsa-miR-520g-4373257 0.045 2.891 hsa-miR-337-3p-002157 0.015 2.851 hsa-miR-516-3p-001149 0.042 0.090 hsa-miR-644-001596 0.042 0.183 hsa-miR-922-002152 0.013 0.286 hsa-miR-193a-3p-4395361 0.017 0.300 hsa-miR-92a-1#-002137 0.038 0.371 hsa-miR-30c-1#-002108 0.004 0.373 hsa-miR-1291-002838 0.017 0.415 hsa-miR-15a-4373123 0.039 0.472

TABLE 7 miRNAs differentially expressed in PD compared to ILBD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (PD/ILBD) hsa-miR-302d-000535 0.004 6.208 hsa-miR-23a-4373074 0.037 4.953 hsa-miR-1290-002863 0.018 3.974 hsa-miR-26a-2#-002115 0.025 3.090 hsa-miR-638-001582 0.005 3.028 hsa-miR-886-3p-4395305 0.049 2.740 hsa-miR-548c-5p-4395540 0.040 2.241 hsa-miR-92a-1#-002137 0.010 2.141 hsa-miR-1224-3P-002752 0.049 0.273 hsa-miR-122-4395356 0.019 0.308 hsa-miR-302a-4378070 0.009 0.357 hsa-miR-424#-002309 0.032 0.380 hsa-miR-486-5p-4378096 0.010 0.409 hsa-miR-541-4395312 0.004 0.455

TABLE 8 miRNAs differentially expressed in PDD compared to ILBD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value fold change (PDD/ILBD) hsa-miR-302d-000535 0.008 5.830 hsa-miR-26a-2#-002115 0.002 4.415 hsa-miR-630-001563 0.046 3.232 hsa-miR-659-001514 0.030 3.229 hsa-miR-641-001585 0.006 2.826 hsa-miR-638-001582 0.011 2.586 hsa-miR-125b-2#-002158 0.044 2.458 hsa-miR-1255B-002801 0.010 2.387 hsa-miR-337-3p-002157 0.048 2.190 hsa-miR-380-5p-000570 0.009 2.058 hsa-miR-202-4395474 0.005 0.260 hsa-miR-873-4395467 0.001 0.279 hsa-miR-767-5p-001993 0.040 0.298 hsa-miR-431-4395173 0.010 0.317 hsa-miR-1224-3P-002752 0.050 0.371 hsa-miR-24-1#-002440 0.034 0.408 hsa-miR-15a-4373123 0.002 0.455 hsa-miR-339-5p-4395368 0.001 0.494

TABLE 9 miRNAs differentially expressed in PDD compared to PD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PDD/PD) dme-miR-7-000268 0.031 4.086 hsa-miR-135b#-002159 0.049 2.608 hsa-miR-641-001585 0.003 2.553 hsa-miR-149#-002164 0.006 2.490 hsa-miR-154#-000478 0.007 2.291 hsa-miR-521-4373259 0.028 2.192 hsa-miR-566-001533 0.035 0.115 hsa-miR-767-5p-001993 0.025 0.214 hsa-miR-202-4395474 0.008 0.321 hsa-miR-548b-5p-4395519 0.025 0.356 hsa-miR-217-4395448 0.011 0.414 hsa-miR-326-4373050 0.042 0.463

TABLE 10 miRNAs Differentially Expressed in Putamen among Different Stage Groups by the Unified Staging System for LBD (p < 0.05; at least 2 fold change). (59 in total) Stages 0 2 3 4 0 29 (9, 20) 24 (11, 13) 10 (3/7) 2 29 (9, 20) 9 (4, 5) 13 (7, 6) 3 24 (11, 13) 9 (4, 5) 7 (3, 4) 4 10 (3/7) 13 (7, 6) 7 (3, 4) *Numbers in parentheses are numbers of (upregulated, downregulated) miRNAs.

TABLE 11 miRNAs differentially expressed in the putamen of Stage II compared to controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage2/0) hsa-miR-1224-3P-002752 0.016 8.494 hsa-miR-767-5p-001993 0.027 5.601 mmu-let-7d#-001178 0.008 3.631 hsa-miR-33b-4395196 0.035 2.762 hsa-miR-516b-4395172 0.033 2.435 hsa-miR-183-4395380 0.006 2.427 hsa-miR-1-4395333 0.031 2.393 hsa-miR-127-5p-4395340 0.048 2.089 hsa-miR-489-4395469 0.001 2.031 hsa-miR-644-001596 0.015 0.157 hsa-miR-1290-002863 0.003 0.250 hsa-miR-1248-002870 0.015 0.254 hsa-miR-638-001582 0.001 0.269 hsa-miR-639-001583 0.017 0.274 hsa-miR-1303-002792 0.025 0.284 hsa-miR-30c-1#-002108 0.000 0.310 hsa-miR-378-000567 0.041 0.318 hsa-miR-26a-2#-002115 0.036 0.326 hsa-miR-1291-002838 0.002 0.328 hsa-miR-616-4395525 0.015 0.342 hsa-miR-922-002152 0.028 0.346 hsa-miR-1244-002791 0.000 0.358 hsa-miR-548c-5p-4395540 0.037 0.366 hsa-miR-92a-1#-002137 0.015 0.385 hsa-miR-497-001043 0.000 0.392 hsa-miR-601-001558 0.012 0.399 hsa-miR-720-002895 0.046 0.430 hsa-miR-1260-002896 0.004 0.452 hsa-miR-635-001578 0.041 0.495

TABLE 12 miRNAs differentially expressed in the putamen of Stage III compared to controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 3/0) hsa-miR-566-001533 0.037 13.485 hsa-miR-1224-3P-002752 0.016 10.196 hsa-miR-98-4373009 0.046 4.805 hsa-miR-767-5p-001993 0.006 3.802 hsa-miR-1-4395333 0.039 3.115 hsa-miR-516b-4395172 0.018 2.728 hsa-miR-33b-4395196 0.049 2.448 hsa-miR-183-4395380 0.017 2.382 hsa-miR-424-4373201 0.017 2.288 hsa-miR-127-5p-4395340 0.033 2.144 hsa-miR-1270-002807 0.049 2.080 hsa-miR-34b-000427 0.031 0.231 hsa-miR-922-002152 0.037 0.286 hsa-miR-30c-1#-002108 0.001 0.323 hsa-miR-106b#-002380 0.000 0.353 hsa-miR-199a-5p-4373272 0.047 0.357 hsa-miR-520D-3P-002743 0.005 0.370 hsa-miR-100#-002142 0.000 0.400 hsa-miR-638-001582 0.022 0.401 hsa-miR-1226#-002758 0.023 0.408 hsa-miR-92a-1#-002137 0.039 0.411 hsa-miR-374b#-002391 0.005 0.436 hsa-miR-497-001043 0.006 0.460 hsa-miR-1260-002896 0.009 0.463

TABLE 13 miRNAs differentially expressed in the putamen of Stage IV compared to controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/0) hsa-miR-518a-3p-4395508 0.027 2.605 hsa-miR-1269-002789 0.043 2.424 hsa-miR-876-3p-4395336 0.003 2.310 hsa-miR-516-3p-001149 0.025 0.095 hsa-miR-644-001596 0.038 0.192 hsa-miR-639-001583 0.022 0.257 hsa-miR-193a-3p-4395361 0.029 0.286 hsa-miR-30c-1#-002108 0.016 0.412 hsa-miR-635-001578 0.033 0.461 hsa-miR-511-4373236 0.014 0.497

TABLE 14 miRNAs differentially expressed in the putamen of Stage III compared to Stage II (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 3/2) hsa-miR-616-4395525 0.029 2.383 hsa-miR-363#-001283 0.036 3.944 hsa-miR-604-001567 0.046 2.567 hsa-miR-639-001583 0.004 4.937 hsa-miR-199a-5p-4373272 0.037 0.287 hsa-let-7f-1#-002417 0.018 0.161 hsa-miR-1201-002781 0.035 0.371 hsa-miR-1301-002827 0.022 0.441 hsa-miR-920-002150 0.040 0.372

TABLE 15 miRNAs differentially expressed in the putamen of Stage IV compared to Stage II (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/2) hsa-miR-1290-002863 0.005 3.942 hsa-miR-1248-002870 0.041 2.939 hsa-miR-638-001582 0.005 2.776 hsa-miR-1303-002792 0.036 2.440 hsa-miR-296-3p-4395212 0.031 2.412 hsa-miR-1255B-002801 0.004 2.314 hsa-miR-378-000567 0.031 2.149 hsa-let-7f-1#-002417 0.008 0.136 hsa-miR-1224-3P-002752 0.014 0.210 hsa-miR-571-001613 0.041 0.224 mmu-let-7d#-001178 0.037 0.386 hsa-miR-1227-002769 0.005 0.486 hsa-miR-511-4373236 0.011 0.497

TABLE 16 miRNAs differentially expressed in the putamen of Stage IV compared to Stage III (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/3) hsa-miR-211-4373088 0.029 50.755 hsa-miR-876-3p-439533

0.003 2.479 hsa-miR-100#-002142 0.005 2.208 hsa-miR-1224-3P-00275

0.008 0.175 hsa-miR-639-001583 0.006 0.190 hsa-miR-363#-001283 0.040 0.248 hsa-miR-873-4395467 0.019 0.434

indicates data missing or illegible when filed

We also identified expressions of 29 miRNAs are correlated with the global Lewy-type asynucleinopathy (LTS) scores; 32 miRNAs are correlated with the degree of depigmentation of the neurons in Substantia Nigra; 33 miRNAs are correlated with the worsening of the motor functions, Unified PD Rating System (UPDRS) (Table 17-20).

TABLE 17 Disease-correlated miRNAs in the Putamen (p < 0.05) By LTS By SN depigmentation By Score* score UPDRS** Positively correlated 15 20 21 Negatively correlated 14 12 16 *LTS score: global Lewy-type α-synucleinopathy (LTS) scores. **UPDRS: Unified PD Rating Scale.

TABLE 18 miRNAs whose expression levels are correlated with Lewy-type a-synucleinopathy (LTS) scores (p < 0.05) miRNAs r hsa-miR-132-4373143 0.505 hsa-miR-212-4373087 0.453 hsa-miR-383-4373018 0.449 hsa-miR-885-5p-4395407 0.425 hsa-miR-132#-002132 0.406 hsa-miR-608-001571 0.401 hsa-miR-183#-002270 0.391 hsa-miR-587-001540 0.375 hsa-miR-1269-002789 0.375 hsa-miR-629-001562 0.370 hsa-let-7c-4373167 0.350 hsa-miR-559-001527 0.349 hsa-miR-129-3p-4373297 0.345 hsa-miR-130b#-002114 0.344 hsa-miR-125a-5p-4395309 0.322 hsa-miR-574-3p-4395460 −0.317 hsa-miR-374a-4373028 −0.320 hsa-miR-720-002895 −0.322 hsa-miR-1275-002840 −0.323 hsa-miR-142-5p-4395359 −0.330 hsa-miR-124-4373295 −0.330 hsa-miR-26b#-002444 −0.331 hsa-miR-1226#-002758 −0.350 hsa-miR-374b#-002391 −0.350 hsa-miR-193a-3p-4395361 −0.364 hsa-miR-590-5p-4395176 −0.379 hsa-miR-660-4380925 −0.388 hsa-miR-339-5p-4395368 −0.458 hsa-miR-106b#-002380 −0.473

TABLE 19 miRNAs whose expression levels are correlated with SN depigmentation scores (p < 0.05) miRNAs r hsa-miR-132-4373143 0.548 hsa-miR-212-4373087 0.444 hsa-miR-1269-002789 0.410 hsa-miR-455-5p-4378098 0.377 hsa-miR-500-4395539 0.376 hsa-miR-139-3p-4395424 0.371 hsa-miR-183#-002270 0.366 hsa-miR-518b-4373246 0.361 hsa-miR-455-3p-4395355 0.348 hsa-miR-629-001562 0.341 hsa-miR-132#-002132 0.340 hsa-miR-23a-4373074 0.337 hsa-miR-335-4373045 0.320 hsa-miR-580-001621 0.315 hsa-miR-383-4373018 0.312 hsa-miR-608-001571 0.312 hsa-miR-548l-002909 0.311 hsa-miR-505#-002087 0.299 hsa-let-7c-4373167 0.297 hsa-miR-129-3p-4373297 0.291 hsa-miR-17-4395419 −0.314 hsa-miR-503-4373228 −0.315 hsa-miR-193a-3p-435361 −0.322 hsa-miR-339-5p-4395368 −0.327 hsa-miR-140-5p-4373374 −0.327 hsa-miR-574-3p-4395460 −0.347 hsa-miR-302a-4378070 −0.361 hsa-miR-10b#-002315 −0.366 hsa-miR-93-4373302 −0.374 hsa-miR-590-5p-4395176 −0.419 hsa-miR-106b#-002380 −0.461 hsa-miR-124-4373295 −0.500

TABLE 20 miRNAs whose expression levels are correlated with UPDRS (p < 0.05) miRNAs r hsa-miR-198-4395384 0.582 hsa-miR-886-3p-4395305 0.528 hsa-miR-450a-4395414 0.525 hsa-miR-593-001547 0.523 hsa-miR-920-002150 0.518 hsa-miR-521-4373259 0.507 hsa-miR-629-4395547 0.501 hsa-miR-517#-001113 0.469 hsa-miR-520c-3p-002400 0.461 hsa-miR-125b-2#-002158 0.461 hsa-miR-27a#-002445 0.456 hsa-miR-559-001527 0.447 hsa-miR-589-001543 0.442 hsa-miR-886-5p-4395304 0.430 hsa-miR-1282-002803 0.430 hsa-miR-1270-002807 0.423 hsa-miR-99a#-002141 0.422 hsa-miR-181a-2#-002317 0.422 hsa-miR-608-001571 0.380 hsa-miR-211-4373088 0.378 hsa-miR-500-001046 0.376 hsa-miR-758-4395180 −0.408 hsa-miR-144-002676 −0.415 hsa-miR-138-4395395 −0.415 hsa-miR-92a-1#-002137 −0.422 hsa-miR-411-4381013 −0.425 hsa-miR-448-4373206 −0.427 hsa-miR-324-5p-4373052 −0.432 hsa-miR-203-4373095 −0.439 hsa-miR-107-4373154 −0.440 hsa-miR-488-4395468 −0.462 hsa-miR-137-4373301 −0.469 hsa-miR-148a-4373130 −0.475 hsa-miR-296-5p-4373066 −0.477 hsa-miR-218-4373081 −0.479 hsa-miR-153-4373305 −0.507 hsa-miR-552-001520 −0.521

These miRNAs may be involved in the pathological changes in the putamen, contributing to the pathogenesis of Lewy body diseases, including PD; and therefore, they may be new therapeutic targets for treatment of PD at different stages of the diseases.

Example 2 CSF miRNAs as Diagnostic Biomarkers for Presymptomatic and Early PD, and Cognitive Impairment in PD

Among the differentially expressed miRNAs in the CSF, we identified 80 miRNAs that are differentially expressed in ILBD, PD and PDD, when compared to normal controls (Tables 21-27; FIG. 3); and 87 miRNAs that are differentially expression in Stages 2, 3, and 4 of Lewy Body diseases when compared to normal controls (Table 28-34; FIG. 4).

TABLE 21 miRNAs Differentially Expressed in CSF Among Different Diagnostic Groups (p < 0.05; at least 2 fold change). (80 in total) Diagnosis Control ILBD PD PDDnoAD Control 35 (25, 10) 27 (15, 12) 15 (13, 2) ILBD 35 (25, 10) 11 (5, 6) 14 (8, 6) PD 27 (15, 12) 11 (5, 6) 20 (8, 12) PDDnoAD 15 (13, 2) 14 (8, 6) 20 (8, 12) *Numbers in parentheses are numbers of (upregulated, downregulated) miRNAs.

TABLE 22 CSF miRNAs differentially expressed in ILBD compared to control (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (ILBD/ctl) hsa-miR-497-001043 0.008 5.645 hsa-miR-126#-000451 0.026 5.413 hsa-miR-9#-002231 0.005 5.231 hsa-miR-1300-002902 0.023 5.129 hsa-miR-143-4395360 0.007 4.804 hsa-miR-374b-4381045 0.027 4.747 hsa-miR-660-4380925 0.005 4.465 hsa-miR-432-001026 0.046 4.313 hsa-miR-31#-002113 0.012 3.647 hsa-miR-324-5p-4373052 0.007 3.367 hsa-miR-99a#-002141 0.024 3.323 hsa-miR-628-3p-002434 0.002 3.318 hsa-miR-652-4395463 0.035 3.011 hsa-miR-1271-002779 0.039 2.775 hsa-miR-26b#-002444 0.016 2.706 hsa-miR-195-4373105 0.000 2.680 hsa-miR-340-4395369 0.031 2.568 hsa-miR-213-000516 0.032 2.524 hsa-miR-29a-4395223 0.003 2.483 hsa-miR-449b-4381011 0.045 2.348 hsa-miR-708-4395452 0.021 2.292 hsa-miR-29c-4395171 0.006 2.248 hsa-miR-218-4373081 0.016 2.223 hsa-miR-488-001106 0.014 2.155 hsa-miR-30e-3p-000422 0.035 2.154 hsa-miR-516-3p-001149 0.040 0.068 hsa-miR-296-5p-4373066 0.048 0.252 hsa-miR-671-3p-4395433 0.021 0.274 hsa-miR-145-4395389 0.016 0.289 hsa-miR-597-4380960 0.012 0.309 hsa-miR-328-4373049 0.025 0.362 hsa-miR-197-4373102 0.039 0.382 hsa-miR-92a-4395169 0.019 0.405 hsa-miR-484-4381032 0.023 0.432 hsa-miR-700-002895 0.044 0.449

TABLE 23 CSF miRNAs differentialiy expressed in PD compared to control (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PD/ctl) hsa-miR-664-002897 0.001 18.334 hsa-miR-590-3P-002677 0.000 5.568 hsa-miR-577-002675 0.033 4.923 hsa-miR-374b-4381045 0.032 4.850 hsa-miR-9#-002231 0.006 4.755 hsa-miR-213-000516 0.001 4.217 rno-miR-7#-001338 0.044 3.712 hsa-miR-340#-002259 0.033 3.561 hsa-miR-497-001043 0.050 3.504 hsa-miR-454-4395434 0.013 3.093 hsa-miR-99a#-002141 0.049 3.050 hsa-miR-191#-002678 0.005 2.907 hsa-miR-1183-002841 0.038 2.387 hsa-miR-29a#-002447 0.030 2.243 hsa-miR-148b#-002160 0.014 2.137 hsa-miR-142-3p-4373136 0.029 0.260 hsa-miR-519a-4395526 0.016 0.318 hsa-miR-511-4373236 0.043 0.319 hsa-miR-486-5p-4378096 0.012 0.428 hsa-miR-92a-4395169 0.046 0.435 hsa-miR-381-4373020 0.037 0.435 hsa-miR-545-4395378 0.010 0.439 hsa-miR-196b-4395326 0.007 0.458 hsa-miR-10b-4395329 0.007 0.463 hsa-miR-98-4373009 0.006 0.465 hsa-miR-18a-4395533 0.003 0.470 hsa-miR-654-3p-4395350 0.008 0.473

TABLE 24 CSF miRNAs differentially expressed in PDDnoAD compared to control (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PDDnoAD/ctl) hsa-miR-1300-002902 0.001 10.044 hsa-miR-664-002897 0.040 6.381 hsa-miR-374b-4381045 0.011 5.426 hsa-miR-1298-002861 0.033 4.569 hsa-miR-135b-4395372 0.003 3.592 hsa-miR-363-4378090 0.002 3.357 hsa-miR-660-4380925 0.032 3.189 hsa-miR-9#-002231 0.029 2.793 hsa-miR-362-5p-4378092 0.033 2.287 hsa-miR-29a-4395223 0.005 2.209 hsa-miR-1264-002799 0.028 2.090 hsa-miR-29a#-002447 0.049 2.073 hsa-miR-29c-4395171 0.003 2.043 hsa-miR-146b-5p-4373178 0.010 0.430 hsa-miR-518a-3p-4395508 0.020 0.372

TABLE 25 CSF miRNAs differentially expressed in PD compared to ILBD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PD/ILBD) hsa-miR-664-002897 0.028 4.750 hsa-miR-720-002895 0.006 3.384 hsa-miR-590-3P-002677 0.028 2.431 hsa-miR-625#-002432 0.033 2.096 hsa-miR-1183-002841 0.032 2.087 hsa-miR-143-4395360 0.026 0.315 hsa-miR-519a-4395526 0.005 0.334 hsa-miR-130a-4373145 0.025 0.414 hsa-miR-130b-4373144 0.037 0.422 hsa-miR-500-4395539 0.041 0.424 hsa-miR-145#-002149 0.036 0.426

TABLE 26 CSF miRNAs differentially expressed in PDDnoAD compared to ILBD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PDDnoAD/ILBD) hsa-miR-886-5p-4395304 0.024 4.734 hsa-miR-886-3p-4395305 0.022 4.189 hsa-miR-125a-3p-4395310 0.011 2.761 hsa-miR-222#-002097 0.040 2.635 hsa-miR-362-5p-4378092 0.006 2.392 hsa-miR-105-4395278 0.048 2.358 hsa-miR-363-4378090 0.036 2.203 hsa-let-7d-4395394 0.032 2.128 hsa-miR-143-4395360 0.011 0.329 hsa-miR-543-002376 0.034 0.430 hsa-miR-628-3p-002434 0.029 0.441 hsa-miR-26b#-002444 0.011 0.460 hsa-miR-146b-5p-4373178 0.035 0.494 hsa-miR-376c-4395233 0.035 0.497

TABLE 27 CSF miRNAs differentially expressed in PDDnoAD compared to PD (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold Change (PDDnoAD/PD) hsa-miR-148b-4373129 0.005 4.709 hsa-miR-886-5p-4395304 0.013 3.754 hsa-miR-500-4395539 0.006 3.002 hsa-miR-886-3p-4395305 0.032 2.889 hsa-miR-519a-4395526 0.045 2.654 hsa-miR-511-4373236 0.035 2.279 hsa-miR-363-4378090 0.037 2.114 hsa-miR-99b-4373007 0.010 2.038 dme-miR-7-000268 0.015 0.118 hsa-miR-1233-002768 0.027 0.229 hsa-miR-409-3p-002332 0.034 0.248 hsa-miR-206-000510 0.027 0.263 hsa-miR-590-3P-002677 0.001 0.275 hsa-miR-213-000516 0.006 0.316 hsa-miR-629-001562 0.041 0.357 hsa-miR-539-4378103 0.037 0.385 hsa-miR-543-002376 0.004 0.407 hsa-miR-720-002895 0.014 0.438 hsa-miR-146b-5p-4373178 0.022 0.453 hsa-miR-136#-002100 0.013 0.465

TABLE 28 miRNAs Differentially Expressed in CSF Among Different Stages by the Unified Staging System for LBD (p < 0.05; at least 2 fold change) (87 in total). Stages 0 2 3 4 0 22 (18, 4) 70 (46, 24) 9 (5, 4) 2 22 (18, 4) 20 (15, 5) 2 (0, 2) 3 70 (46, 24) 20 (15, 5) 29 (7, 22) 4 9 (5, 4) 2 (0, 2) 29 (7, 22) *Numbers in parentheses: numbers of (upregulated, downregulated) miRNAs.

TABLE 29 CSF miRNAs differentially expressed in Stage II compared to controls (corrected p values < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 2/0) hsa-miR-664-002897 0.035 7.145 hsa-miR-126#-000451 0.041 6.557 hsa-miR-374b-4381045 0.023 5.102 hsa-miR-31-4395390 0.007 4.549 hsa-miR-1300-002902 0.032 3.927 hsa-miR-577-002675 0.032 3.690 hsa-miR-9#-002231 0.006 3.655 hsa-miR-497-001043 0.047 3.493 hsa-miR-660-4380925 0.025 3.394 hsa-miR-652-4395463 0.018 3.212 hsa-miR-31#-002113 0.032 2.830 hsa-miR-340-4395369 0.037 2.768 hsa-miR-200b-4395362 0.034 2.719 hsa-miR-26b#-002444 0.034 2.437 hsa-miR-135b-4395372 0.049 2.391 hsa-miR-195-4373105 0.003 2.243 hsa-miR-628-3p-002434 0.027 2.081 hsa-miR-29a#-002447 0.048 2.002 hsa-miR-671-3p-4395433 0.023 0.282 hsa-miR-142-3p-4373136 0.038 0.348 hsa-miR-145-4395389 0.043 0.407 hsa-miR-597-4380960 0.047 0.408

TABLE 30 CSF miRNAs differentiallly expressed in Stage III compared to controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 3/0) hsa-miR-661-001606 0.008 35.493 hsa-miR-1300-002902 0.002 14.271 hsa-miR-497-001043 0.000 10.198 hsa-miR-664-002897 0.023 7.823 hsa-miR-577-002675 0.013 7.754 hsa-miR-9#-002231 0.003 7.611 hsa-miR-769-5p-001998 0.020 6.804 hsa-miR-374b-4381045 0.014 5.960 hsa-miR-31#-002113 0.004 5.909 hsa-miR-99a#-002141 0.002 5.850 hsa-miR-638-001582 0.014 5.810 hsa-miR-660-4380925 0.005 5.749 hsa-miR-1298-002861 0.037 5.389 hsa-miR-592-001546 0.018 4.821 rno-miR-29c#-001818 0.011 4.648 hsa-miR-324-5p-4373052 0.001 4.592 hsa-miR-590-3P-002677 0.001 4.436 hsa-miR-213-000516 0.004 4.032 hsa-miR-1271-002779 0.005 3.965 hsa-miR-9-4373285 0.034 3.796 hsa-miR-622-001553 0.017 3.549 hsa-miR-340#-002259 0.037 3.533 hsa-miR-151-5P-002642 0.035 3.373 hsa-miR-628-3p-002434 0.006 3.265 hsa-miR-29a-4395223 0.001 3.218 hsa-miR-143-4395360 0.046 3.190 hsa-miR-135b-4395372 0.013 3.045 hsa-miR-652-4395463 0.031 2.942 hsa-miR-129-3p-4373297 0.045 2.926 hsa-miR-218-4373081 0.004 2.880 hsa-miR-454-4395434 0.034 2.870 hsa-miR-340-4395369 0.028 2.834 hsa-miR-29c-4395171 0.001 2.802 hsa-miR-1226#-002758 0.030 2.670 hsa-miR-30a-3p-000416 0.012 2.617 hsa-miR-639-001583 0.042 2.574 hsa-miR-195-4373105 0.000 2.533 hsa-miR-191#-002678 0.007 2.389 hsa-miR-29a#-002447 0.030 2.254 hsa-miR-148b#-002160 0.020 2.173 hsa-miR-192-4373108 0.028 2.137 hsa-miR-148a-4373130 0.003 2.135 hsa-miR-548l-002909 0.036 2.055 hsa-miR-1264-002799 0.004 2.052 hsa-miR-19b-4373098 0.014 2.013 hsa-miR-27a#-002445 0.021 2.007 hsa-miR-483-3p-002339 0.005 0.081 hsa-miR-1260-002896 0.026 0.122 hsa-miR-296-5p-4373066 0.019 0.140 hsa-miR-145-4395389 0.001 0.161 hsa-miR-671-3p-4395433 0.009 0.166 hsa-miR-125a-5p-4395309 0.011 0.243 hsa-miR-197-4373102 0.005 0.257 hsa-miR-92a-4395169 0.002 0.284 hsa-miR-597-4380960 0.022 0.292 hsa-miR-328-4373049 0.001 0.305 hsa-miR-484-4381032 0.002 0.319 hsa-miR-125b-4373148 0.009 0.363 hsa-miR-345-4395297 0.019 0.413 hsa-miR-10b-4395329 0.008 0.461 hsa-miR-365-4373194 0.039 0.463 hsa-miR-98-4373009 0.008 0.464 hsa-miR-545-4395378 0.020 0.468 hsa-miR-28-3p-4395557 0.000 0.469 hsa-miR-25-4373071 0.006 0.472 hsa-miR-654-3p-4395350 0.009 0.474 hsa-miR-486-5p-4378096 0.033 0.475 hsa-miR-196b-4395326 0.014 0.482 hsa-miR-193a-5p-4395392 0.050 0.486 hsa-miR-199a-5p-4373272 0.040 0.497

TABLE 31 CSF miRNAs differentially expressed in Stage IV compared to controls (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/0) hsa-miR-664-002897 0.0267 6.6111 hsa-miR-374b-4381045 0.0298 4.2124 hsa-miR-1300-002902 0.0443 3.9213 hsa-miR-9#-002231 0.0246 2.8799 hsa-miR-191#-002678 0.0377 2.2135 hsa-miR-142-3p-4373136 0.0103 0.2291 hsa-miR-197-4373102 0.0326 0.4014 hsa-miR-518a-3p-4395508 0.0247 0.4188 hsa-miR-320B-002844 0.0210 0.4882

TABLE 32 CSF miRNAs differentially expressed in Stage III compared to Stage II (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 3/2) hsa-miR-661-001606 0.014 10.926 hsa-miR-638-001582 0.018 4.388 hsa-miR-144#-002148 0.002 4.147 hsa-miR-566-001533 0.010 3.230 hsa-miR-622-001553 0.022 3.205 hsa-miR-497-001043 0.031 2.919 hsa-miR-663B-002857 0.024 2.748 hsa-miR-1226#-002758 0.023 2.555 hsa-miR-30a-3p-000416 0.002 2.417 hsa-miR-324-5p-4373052 0.040 2.290 hsa-miR-590-3P-002677 0.042 2.244 rno-miR-29c#-001818 0.044 2.193 hsa-miR-148a-4373130 0.015 2.140 hsa-miR-31#-002113 0.023 2.088 hsa-miR-218-4373081 0.029 2.077 hsa-miR-483-3p-002339 0.011 0.142 hsa-miR-125b-4373148 0.001 0.251 hsa-miR-125a-5p-4395309 0.013 0.272 hsa-miR-145-4395389 0.015 0.395 hsa-miR-130b-4373144 0.047 0.448

TABLE 33 CSF miRNAs differentially expressed in Stage IV compared to Stage II (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/2) hsa-miR-181a-4373117 0.022 0.436 hsa-miR-338-3p-4395363 0.049 0.459

TABLE 34 CSF miRNAs differentially expressed in Stage IV compared to Stage III (corrected p value < 0.05, fold change > 2 folds) miRNAs p-value Fold change (Stage 4/3) hsa-miR-483-3p-002339 0.041 5.103 hsa-miR-145-4395389 0.001 4.830 hsa-miR-296-5p-4373066 0.033 4.172 hsa-miR-125a-5p-4395309 0.010 3.893 hsa-miR-671-3p-4395433 0.022 3.134 hsa-miR-125b-4373148 0.001 2.949 hsa-miR-484-4381032 0.014 2.231 hsa-miR-661-001606 0.010 0.142 hsa-miR-497-001043 0.007 0.216 hsa-miR-1291-002838 0.004 0.240 hsa-miR-638-001582 0.013 0.244 hsa-miR-31#-002113 0.004 0.245 hsa-miR-378-002243 0.013 0.267 hsa-miR-1271-002779 0.002 0.267 hsa-miR-219-2-3p-4395501 0.033 0.272 hsa-miR-622-001553 0.010 0.289 hsa-miR-99a#-002141 0.007 0.294 hsa-miR-144#-002148 0.024 0.324 hsa-miR-769-5p-001998 0.037 0.325 hsa-miR-6638-002857 0.025 0.361 hsa-miR-1226#-002758 0.011 0.363 hsa-miR-142-3p-4373136 0.033 0.379 hsa-miR-151-5P-002642 0.001 0.382 hsa-miR-3208-002844 0.000 0.413 hsa-miR-12558-002801 0.044 0.417 hsa-miR-660-4380925 0.006 0.439 hsa-miR-192-4373108 0.045 0.479 hsa-miR-218-4373081 0.026 0.485 hsa-miR-30a-3p-000416 0.026 0.493

We also identified expressions of 26 miRNAs are correlated with the global Lewy-type asynucleinopathy (LTS) scores; 36 miRNAs are correlated with the degree of depigmentation of the neurons in Substantia Nigra; 40 miRNAs are correlated with the worsening of the motor functions, Unified PD Rating System (UPDRS) (Tables 35-38).

TABLE 35 Disease-correlated miRNAs in CSF (p < 0.05) By LTS By SN depigmentation By Score* score UPDRS** Positively correlated 23 19 9 Negatively correlated 3 17 31 *LTS score: global Lewy-type α-synucleinopathy (LTS) scores. **UPDRS: Unified PD Rating Scale.

TABLE 36 CSF miRNAs whose expression levels are correlated with Lewy-type a-synucleinopathy (LTS) scores (p < 0.05) miRNAs r hsa-miR-664-002897 0.519 hsa-miR-148b#-002160 0.496 hsa-miR-29a#-002447 0.453 hsa-miR-592-001546 0.403 hsa-miR-363-4378090 0.400 hsa-miR-135b-4395372 0.397 hsa-miR-135a-4373140 0.390 hsa-miR-1298-002861 0.389 hsa-miR-34a#-002316 0.389 hsa-miR-374b-4381045 0.384 rno-miR-7#-001338 0.381 hsa-miR-590-3P-002677 0.379 rno-miR-29c#-001818 0.371 hsa-miR-1264-002799 0.371 hsa-miR-22#-002301 0.358 hsa-miR-30a-3p-000416 0.353 hsa-miR-650-001603 0.346 hsa-miR-340-4395369 0.338 hsa-miR-454-4395434 0.336 hsa-miR-767-3p-001995 0.333 hsa-miR-101#-002143 0.329 hsa-miR-190-4373110 0.328 hsa-miR-213-000516 0.320 hsa-miR-486-5p-4378096 −0.373 hsa-miR-196b-4395326 −0.435 hsa-miR-518a-3p-4395508 −0.439

TABLE 37 CSF miRNAs whose expression levels are correlated with SN depigmentation scores (p < 0.05) miRNAs r hsa-miR-363-4378090 0.443 hsa-miR-374b-4381045 0.395 hsa-miR-184-4373113 0.339 hsa-miR-135b-4395372 0.325 hsa-miR-628-5p-4395544 0.323 hsa-miR-374a-4373028 0.309 hsa-miR-331-3p-4373046 0.294 hsa-miR-217-4395448 0.289 hsa-miR-590-5p-4395176 0.286 hsa-miR-664-002897 0.285 hsa-let-7a-4373169 0.279 hsa-miR-26b-4395167 0.272 hsa-let-7f-4373164 0.263 hsa-miR-362-5p-4378092 0.263 hsa-miR-455-3p-4395355 0.262 hsa-miR-449b-4381011 0.262 hsa-miR-99a-4373008 0.255 hsa-miR-135a-4373140 0.254 hsa-miR-26a-4395166 0.253 hsa-miR-218-2#-002294 −0.227 hsa-miR-139-3p-4395424 −0.232 hsa-miR-143#-002146 −0.246 hsa-miR-185-4395382 −0.249 hsa-miR-630-001563 −0.255 hsa-miR-519a-4395526 −0.255 hsa-miR-376a#-002127 −0.260 hsa-let-7c-4373167 −0.263 hsa-miR-133b-4395358 −0.271 hsa-miR-145#-002149 −0.282 hsa-miR-181a-4373117 −0.299 hsa-miR-182#-000483 −0.308 hsa-miR-200a#-001011 −0.313 hsa-miR-197-4373102 −0.315 hsa-miR-146a#-002163 −0.318 hsa-miR-142-3p-4373136 −0.354 hsa-miR-518a-3p-4395508 −0.363

TABLE 38 CSF miRNAs whose expression levels are correlated with UPDRS (p < 0.05) miRNAs r hsa-miR-101#-002143 0.572 hsa-miR-222#-002097 0.565 hsa-miR-626-001559 0.555 hsa-miR-29a#-002447 0.503 hsa-miR-1238-002927 0.469 hsa-miR-1260-002896 0.453 hsa-miR-363#-001283 0.393 hsa-miR-1244-002791 0.388 hsa-miR-567-001534 0.385 hsa-miR-101-4395364 −0.230 hsa-miR-218-4373081 −0.233 hsa-miR-27a#-002445 −0.234 hsa-miR-330-3p-4373047 −0.236 hsa-miR-30e-3p-000422 −0.237 hsa-miR-29b-2#-002166 −0.239 hsa-miR-411-4381013 −0.241 hsa-miR-875-5p-002203 −0.242 hsa-miR-337-5p-4395267 −0.244 hsa-miR-130a-4373145 −0.245 hsa-miR-454#-001996 −0.257 dme-miR-7-000268 −0.261 hsa-miR-19b-1#-002425 −0.263 hsa-miR-32-4395220 −0.263 hsa-miR-137-4373301 −0.265 hsa-miR-122-4395356 −0.268 hsa-miR-518a-3p-4395508 −0.269 hsa-miR-219-5p-4373080 −0.274 hsa-let-7b-4395446 −0.278 hsa-miR-655-4381015 −0.279 hsa-miR-1264-002799 −0.282 hsa-miR-1262-002852 −0.289 hsa-miR-518b-4373246 −0.290 hsa-miR-152-4395170 −0.292 hsa-miR-338-3p-4395363 −0.294 hsa-miR-520D-3P-002743 −0.316 hsa-miR-488-4395468 −0.354 hsa-miR-551b-4380945 −0.375 hsa-miR-625-4395542 −0.382 hsa-miR-519a-4395526 −0.419 hsa-miR-21-4373090 −0.429

More importantly, we identified a series of miRNA signatures as first generation of diagnostic biomarkers for PD and the progression of PD:

1) miRNA signature composed by 14 miRNAs that distinguishes ILBD to normal controls, which may be used as a diagnostic biomarker for pre-symptomatic PD (FIG. 5): miR-516, miR-191#, miR-449b, miR-497, miR-628, miR-213, miR-23a, miR-29#, miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, miR-99#;

2) miRNA signature composed of 13 miRNAs that distinguishes normal control subjects from PD, which may be used as a diagnostic biomarker for diagnosis of early PD (FIG. 6): miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p;

3) miRNA signature composed by 6 miRNAs that distinguishes ILBD from PD, which may change from asymptomatic Lewis Body disease to symptomatic PD, and may be used as a diagnostic biomarker for diagnosis of early PD (FIG. 7): miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603;

4) miRNA signature composed by 8 miRNAs that distinguish clinically normal subjects (including normal control and ILBD subjects), which may also be used early diagnostic biomarkers for PD (FIG. 8): miR-590-3p, miR-664, miR-519a, miR-340*, miR-720, miR-142-3p, miR-185, and miR-213; and

5) miRNA signature composed by 7 miRNAs which distinguish PD from PD with dementia, and may be used as a diagnostic biomarker for cognitive impairment in PD patients (FIG. 9): miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206 and miR-629.

Example 3 miR-212 and miR-132 Play Important Roles in Pathogenesis of PD and are Novel Therapeutic Targets for Treatment of PD and L-Dopa-Induced Dyskinesia

Among the miRNAs in the putamen, which are correlated with the progression of disease, miR-212 and miR-132 are highly expressed the putamen, and are significantly increased (˜1.6 fold) in the putamen of PD cases, compared to controls (FIG. 8). miR-212 and miR-132 are clustered in intron 1 of a highly-conserved, non-coding RNA gene on human chromosome 1p13.3, and have high sequence homology with the same seed sequences, therefore, share most of their downstream targets and functions (FIG. 10). Interestingly, mature miR-132 is expressed at a much higher level than miR-212 in the putamen (>60 fold), suggesting that miR-132 plays a major role in the putamen.

Correlation study with all cases regardless of their clinical diagnosis showed that levels of miR-212 and miR-132 expression in the putamen are significantly correlated to the global Lewy-type α-synucleinopathy (LTS) scores and the degree of substantia nigra (SN) pigmented neuron loss score (FIG. 11), with the highest correlation scores among all differentially expressed miRNAs, strongly suggesting that expression of miR-212 and/or miR-132 correlates with SN neurodegeneration and PD pathology, and that misregulation of miR-212 and/or miR-132 are involved in PD. Multiple lines of evidence support that miR-212 and/or miR-132 play important roles in pathogenesis of PD and are novel therapeutic targets for treatment of PD and L-Dopa-induced dyskinesia:

1) Our target prediction analysis showed that Nurr1, a member of nuclear receptor superfamily, is a predicted target of miR-212 and/or miR-132. Nurr1 is essential for the differentiation and survival of nigral dopaminergic neurons5-7. Mutations are associated with PD. Therefore, miR-212 and/or miR-132 may regulate the function and survival of dopaminergic (DA) neurons by modulating Nurr1.

2) Our functional annotation analysis of predicted target genes of miR-212 and miR-132 revealed significant enrichment of genes involved in neurotrophin signaling (p=0.01), MAPK signaling (p=0.008) and longterm potentiation (p=0.02), consistent with reports that miR-212 and miR-132 are neurotrophin- and activity-regulated miRNAs and are important for neural plasticity.

3) NMDA receptors (NMDA-Rs) are shown to mediate neurotrophin- and activity-induced induction of miR-212 and miR-132. In PD, depletion of nigrostriatal DA results in relative glutamatergic overactivity in the striatum, which plays central roles in PD as well as L-dopa induced dyskinesia. Therefore, upregulation of miR-212 and/or miR-132 in PD putamen may be a result of loss of dopamine input and relative overactivity through NMDA-R;

4) miR-212 and miR-132 are shown to target Methyl CpG-binding protein-2 (MeCP2), which promote the expression of brain-derived neurotrophic factor (BDNF), a potent trophic factor for DA neurons, which has been shown to be decreased in substantia nigra (SN) and striatum PD patients and animal models. Therefore, increased of expression of miR-212 and/or miR-132 may inhibit the expression of MeCP2 and BDNF, and contribute to the pathogenesis of PD;

5) miR-212 and miR-132 are shown to promote NF-κB- and p53-activation through repression of silent information regulator 1 (SIRT1), a NAD+-dependent deacetylase, which deacetylates and inactivates p53 and the p65 subunit of NF-κB29-32. Increased p53 and NF-κB activation in SN and striatum promote cellular senescence and apoptosis. Therefore, miR-212 and/or miR-132 may contribute to nigrostriatal neurodegeneration through modulating NF-κB and p53 activation.

Collectively, these data suggest that upregulation of miR-212 and/or miR-132 in the putamen may be a result of loss of nigrostriatal DA and overactivation of NMDA-Rs; miR-212 and/or miR-132 may mediate NMDAR overactivation-induced excitotoxicity and play important roles in the pathogenesis of PD through argeting MeCP2, SIRT1 and Nurr1, modulating Nurr1-, BDNF-, NF-κB- and p53-involved pathogenetic pathways. Therefore, prevention of loss of DA innervation-resulted upregulation of miR-212 and/or miR-132 may be neuroprotective for the nigrostriatal system and prevent the development of PD. miR-212 and/or miR-132 may be novel therapeutic targets for the treatment of PD.

The above Figures and disclosure are intended to be illustrative and not exhaustive. This description will suggest many variations and alternatives to one of ordinary skill in the art. All such variations and alternatives are intended to be encompassed within the scope of the attached claims. Those familiar with the art may recognize other equivalents to the specific embodiments described herein which equivalents are also intended to be encompassed by the attached claims. 

1. A method of identifying a subject at risk of developing or having Parkinson's Disease (PD), the method comprising: a) isolating a biological sample from the subject; b) detecting a level of expression in a miRNA signature, the miRNA signature comprising at least one miRNA of SEQ ID NOs: 1-283 in the biological sample from the subject; c) comparing the level of expression in the sample to a level of expression in a reference, wherein an increased or decreased level of expression in the sample compared to the level of expression in the reference identifies the subject as having PD or who is at risk of developing PD.
 2. The method of claim 1, wherein the reference is a sample from a normal, healthy subject.
 3. (canceled)
 4. The method of claim 1, wherein an increased or decreased level of expression of at least one miRNA of SEQ ID NOs: 204, 110, 192, 200, 244, 21, 134, 144, 198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b, miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#), or a combination thereof, is indicative of pre-symptomatic PD.
 5. The method of claim 1, wherein the miRNA signature comprises SEQ ID NOs: 204, 110, 192, 200, 244, 21, 134, 144, 198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b, miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#) and the miRNA signature is indicative of pre-symptomatic PD.
 6. The method of claim 1, wherein an increased or decreased level of expression of at least one miRNA of SEQ ID NOs: 1-13 (miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p) or a combination thereof, is indicative of early PD.
 7. The method of claim 1, wherein the miRNA signature comprises SEQ ID NOs: 1-13, (miR-590-3p, miR-213, miR-9#, miR-191#, miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p) and the miRNA signature is indicative of early PD.
 8. The method of claim 1, wherein an increased or decreased level of expression of at least one miRNA of SEQ ID NOs: 14-21 (miR-590-3p, miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185, and miR-213) or a combination thereof, is indicative of early PD.
 9. The method of claim 1, wherein the mRNA signature comprises SEQ ID NOs: 14-21 (miR-590-3p, miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185, and miR-213) and the miRNA signature is indicative of early PD.
 10. The method of claim 1, wherein an increased or decreased level of expression of at least one miRNA of SEQ ID NOs: 22-27 (miR-664, miR-1285, miR-1183, miR-143, miR-519a, mild-803) or a combination thereof, is indicative of early PD.
 11. The method of claim 1, wherein the miRNA signature comprises SEQ ID NOs: 22-27 (miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603) and the miRNA signature is indicative of early PD.
 12. The method of claim 1, wherein an increased or decreased level of expression of at least one miRNA of SEQ ID NOs: 28-34 (miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629) or a combination thereof, is indicative of cognitive impairment of PD.
 13. The method of claim 1, wherein the miRNA signature comprises SEQ ID NOs: 28-34 (miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629) and the miRNA signature is indicative of cognitive impairment of PD.
 14. A pharmaceutical composition comprising an oligonucleotide that down-regulates the over-expression of at least one miRNA of SEQ ID NOs: 1-283, wherein said oligonucleotide is: a) complementary to the nucleotide sequence of at least one of SEQ ID NOs: 1-283, or b) hybridizes under stringent conditions to a nucleotide sequence of at least one of SEQ ID NOs: 1-283.
 15. The pharmaceutical composition of claim 14, wherein the oligonucleotide is chemically modified.
 16. The pharmaceutical composition of claim 14, wherein the oligonucleotide is 10-50 nucleotides in length.
 17. A method for treating, preventing, or reducing the risk of PD associated with aberrant expression of a miRNA in a cell, tissue, or animal, the method comprising contacting the cell, tissue, or animal with the pharmaceutical composition according to claim
 14. 18. (canceled)
 19. The method of claim 17, where the aberrant expression is up-regulation of the miRNA.
 20. The method of claim 17, where the oligonucleotide is capable of reducing expression of the mRNA by at least 50%.
 21. A kit for performing measurement of a mRNA signature, the miRNA signature comprising at least one mRNA of SEQ ID NOs: 1-283, wherein the kit comprises reagents for measuring an expression level of at least one mRNA of SEQ ID NOs: 1-283.
 22. The kit of claim 21, wherein the kit comprises reagents for measuring SEQ ID NOs: 204, 110, 192, 200, 244, 21, 134, 144, 198, 234, 143, 261, 255, and 179 (miR-516-3p, miR-191#, miR-449b, miR-497, miR-628-3p, miR-213, miR-23a, miR-29a#, miR-488, miR-597, miR-296-5p, miR-671-3p, miR-654-3p, and miR-99a#).
 23. The kit of claim 21, wherein the kit comprises reagents for measuring SEQ ID NOs: 1-13 (miR-590-3p, miR-213, miR-9#, miR-191#₅ miR-497, miR-664, miR-99a#, miR-1183, miR-340#, miR-628-3p, miR-7#, miR-29a#, miR-142-3p).
 24. The kit of claim 21, wherein the kit comprises reagents for measuring SEQ ID NOs: 14-21 (miR-590-3p, miR-664, miR-519a, miR-340#, miR-720, miR-142-3p, miR-185, and miR-213).
 25. The kit of claim 21, wherein the kit comprises reagents for measuring SEQ ID NOs: 22-27 (miR-664, miR-1285, miR-1183, miR-143, miR-519a, miR-603).
 26. The kit of claim 21, wherein the kit comprises reagents for measuring SEQ ID NOs: 28-34 (miR-590-3p, miR-213, miR-409-3p, miR-500, miR-7, miR-206, and miR-629). 